1. Field of the Invention
This invention relates to the use of peptides derived from herpesvirus proteins to improve the specificity of enzyme immunoassays for the detection of herpesvirus-specific IgM antibody.
2. Discussion of the Art
Diagnosis of herpesvirus infection is typically accomplished by serological methods, i.e., demonstration of the appearance of antibodies to herpesvirus antigens in a previously seronegative subject. Herpesvirus IgM antibodies can be a sensitive and specific indicator of primary herpesvirus infection in immunocompetent subjects. However, its detection varies widely because of low levels of IgM antibodies produced in some patients.
A problem with assays utilizing herpesvirus antigens for detecting herpesvirus-specific IgM antibodies is that the assays are so sensitive that low levels of herpesvirus-specific IgM antibodies that are present in individuals of low risk are frequently detected. In other words, these assays indicate too many false positive results. The diagnosis of recent herpesvirus infection, e.g., 0 to 8 months, is questionable because the low level of IgM antibodies present in individuals experiencing an asymptomatic reactivation from a previous herpesvirus infection may be detected. If the level of herpesvirus antigen in the reagent is reduced to improve the specificity of the assay, the sensitivity of the assay is also reduced. In other words, it is undesirable to obtain a positive result in asymptomatic patients who have not had an infection within the previous eight months. Accordingly, it would be desirable to provide an assay for herpesvirus that not only detects IgM antibodies indicative of recent infection, but that also fails to detect IgM antibodies that are not indicative of recent infection.
Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus in human beings. It is rarely pathogenic in healthy adults but is associated with several diseases in immunocompromised individuals (such as persons infected with HIV and transplant recipients). Furthermore, HCMV is the most common cause of congenital infection in humans. Intrauterine primary infections are second only to Down's syndrome as a known cause of mental retardation. Less severe complications result from secondary infections. Because infections are either asymptomatic or accompanied by symptoms that are not specific of HCMV (such as fever and leukopenia), laboratory techniques are the sole means of diagnosing acute HCMV infection. Diagnosis of HCMV infection can be obtained by direct demonstration of the virus or virus components in pathological materials or indirectly through serology. Diagnosis of primary HCMV infection is accomplished exclusively by serological methods, i.e. demonstration of the appearance of antibodies to HCMV in a previously seronegative subject. HCMV-specific IgM is a sensitive and specific indicator of primary HCMV infection in immunocompetent subjects, and it is very often produced during active viral reactivation in transplant recipients. However its detection varies widely and a very poor agreement has been found among the results obtained with different commercial kits.
European Patent Application 262531 discloses immunogenic portions of HCMV structural phosphoprotein of 150 kD, encoded by the gene localized in the Hind III-Y/N fragment of the viral genome; according to that European Patent Application, such immunogenic portions of pp150 are encoded, in particular, by an EcoRI-PstI fragment of approximately 1.5 kb, localized inside the region of EcoRI-Y fragment from HCMV genome of AD169 strain. Subsequent and more exhaustive studies have however shown the disclosure of European Patent Application 262531 to be incorrect, in that protein pp150 is shown to be encoded by UL32, is shown to have a length much greater than 1.5 kb and, finally, is shown to be quite outside any such EcoRI-PstI fragment, as may be defined within the EcoRI-Y fragment of the AD169 strain. Furthermore, the EcoRI-Y fragment is wholly outside of Hind III-Y/N fragment.
According to U.S. Ser. No. 08/765,856, filed Dec. 27, 1996 (hereinafter "U.S. Ser. No. 08/765,856"), there is a correlation between the immunogenic properties shown by the proteic material referred to in the aforementioned European patent application (which, however, providing an incorrect identification of the polypeptide, causes it to remain substantially undetermined) and the inclusion into the peptide of epitope A1C2, encoded bag UL32 nucleotides 1783 to 1842, running 5'.fwdarw.43', i.e., of the region corresponding to amino acids 595 to 614 of ppUL32, whose identification is posterior (Novak J. P. et al. 1991. Mapping of serologically relevant regions of human cytomegalovirus phosphoprotein pp150 using synthetic peptides. J. Gen. Virol. 72; 1409-1413).
U.S. Ser. No. 08/765,856 discloses a recombinant proteic material, in particular, a fusion protein, capable of binding by immunoreaction with antibodies produced against human Cytomegalovirus, in particular, both IgM and IgG, capable of being used as an antigen for detecting such antibodies in the relevant serologic tests, with substantially the same efficacy as the virus and/or of infected cell lyses. U.S. Ser. No. 08/765,856 further discloses diagnostic reagents and kits derived from that proteic material.
U.S. Ser. No. 08/765,856 discloses recombinant antigens, in particular fusion proteins, to detect Cytomegalovirus-specific IgM in human sera by enzyme immunoassay. U.S. Ser. No. 08/765,856 further discloses diagnostic, reagents and kits derived from those proteic materials.
According to U.S. Ser. No. 08/765,856, there is provided a recombinant proteic material, capable of binding with antibodies against human Cytomegalovirus (HCMV), characterized in that it consists of a fusion protein comprising a first region, carrying at least part of the sequence between aa 202 and aa 434, inclusive, read in 5'.fwdarw.3' direction, of protein pp52, a second region, carrying at least a part of the sequence between aa 1006 and aa 1048, inclusive, read in 5'.fwdarw.3' direction, of pp150, and a third region, carrying at least a part of the sequence between aa 595 and 614 inclusive, in 5'.fwdarw.3' direction, of pp150. These regions are capable of being variably arranged with respect to one another within the fusion protein.
These regions preferably comprise the sequences of pp52 and pp150 in full, and, according to a preferred embodiment, a fusion protein according to U.S. Ser. No. 08/765,856 comprises in a sequential manner, running 5'.fwdarw.3': the first region, immediately downstream wherefrom there is placed the third region, and then the second region, downstream from the third region, a bridge sequence being inserted between the third and the second regions. The bridge sequence consists, running 5'.fwdarw.3', of the following series of aminoacids: lys leut gln glu phe. See SEQ ID NO: 7.
U.S. Ser. No. 08/765,856 further provides for a reagent for diagnosing HCMV infection by serological methods comprising a fusion protein as defined previously. In particular, the diagnostic reagent comprises a fusion protein, whereof at least part of the amino acids is encoded by the nucleotide sequence shown in the sequence listing as SEQ ID NO: 1, read from nucleotide 001 to nucleotide 900.
U.S. Ser. No. 08/765,856 also relates to diagnostic reagents for direct detection of HCMV through serology, comprising at least one monospecific polyclonal serum, or monoclonal antibodies (MaB), directed against the fusion protein.
According to another aspect of U.S. Ser. No. 081765,856, there is provided a mixture of recombinant antigens to detect Cytomegalovirus-specific IgM in human sera by enzyme immunoassay, the mixture being characterized in that it includes, in combination:
(i) a first fusion protein comprising: a first region, carrying an amino acid sequence (H10) corresponding to at least part of the sequence between a2L 202 and aa 434, inclusive, read in 5'.fwdarw.3' direction, of viral protein pp52; a second region, carrying an amino acid sequence (F3) corresponding to at least part of the sequence between aa 1006 to aa 1048, inclusive, read in 5'.fwdarw.3' direction, of viral protein pp150; and a third region, carrying an amino acid sequence (A1C2) corresponding to at least part of the sequence between aa 595 and aa 614 inclusive, read in 5'.fwdarw.3' direction, of the same viral protein pp150; which regions are capable of being variably arranged with respect to one another within the first fusion protein; PA1 (ii) a second fusion protein comprising an immunogen region consisting in a sequence of amino acids corresponding to at least part of the sequence between aa 297 to aa 510, inclusive, read in 5'.fwdarw.3' direction, of the major matrix protein pp65 encoded by the viral gene UL83; and PA1 (iii) a third fusion protein comprising an immunogen region consisting in a sequence of amino acids corresponding to at least part of the sequence between aa 117 to aa 373, read in 5'.fwdarw.3' direction, of the viral assembly protein pp38 encoded by the viral gene UL80a. PA1 (i) a first fusion protein comprising: a first region, carrying an amino acid sequence (H10) corresponding to at least part of the sequence between aa 202 and aa 434, inclusive, read in 5'.fwdarw.3' direction, of viral protein pp52; a second region, carrying an amino acid sequence (F3) corresponding to at least part of the sequence between aa 1006 to aa 1048, inclusive, read in 5'.fwdarw.3' direction, of viral pp150; and a third region, carrying an amino acid sequence (A1C2) corresponding to at least part of the sequence between aa 595 and 614 inclusive, read in 5'.fwdarw.3' direction, of the same viral protein pp150; which regions are capable of being variably arranged with respect to one another within the first fusion protein; PA1 (ii) a second fusion protein comprising an immunogen region consisting in a sequence of amino acids corresponding to at least part of the sequence between aa 297 to aa 510, inclusive, read in 5'.fwdarw.3' direction, of the major matrix protein pp65 encoded by the viral gene UL83; and PA1 (iii) a third fusion protein comprising an immunogen region consisting in a sequence of amino acids corresponding to at least part of the sequence between aa 117 to aa 373, read in 5'.fwdarw.3' direction, of the viral assembly protein pp38 encoded by the viral gene UL80a. PA1 (a) providing a solid phase containing at least a portion of a herpesvirus antigen; PA1 (b) introducing a patient specimen to the solid phase of step (a); PA1 (c) introducing at least one peptide capable of specifically binding IgM antibodies to herpesvirus to the solid phase of step (a); PA1 (d) allowing IgM antibodies to herpesvirus to specifically bind to the portion of antigen of herpesvirus and the peptide capable of specifically binding IgM antibodies to herpesvirus; and PA1 (e) determining the level of IgM antibodies to herpesvirus in the patient specimen. PA1 (a) microparticles coated with at least a portion of herpesvirus antigen, preferably suspended in a diluent containing at least one peptide capable of specifically binding IgM antibodies to herpesvirus antigen; PA1 (b) IgM conjugate in a diluent; and PA1 (c) buffered diluent containing a blocking agent. PA1 (a) providing a solid phase containing at least a portion of human cytomegalovirus, preferably the pp150 protein (hereinafter "pp 150"); PA1 (b) introducing a patient specimen to the solid phase of step (a); PA1 (c) introducing at least one peptide capable of specifically binding IgM antibodies to human cytomegalovirus to the solid phase of step (a); PA1 (d) allowing IgM antibodies to human cytomegalovirus to specifically bind to the portion of human cytomegalovirus, preferably pp150, and the peptide capable of specifically binding IgM antibodies to human cytomegalovirus; and PA1 (e), determining the level of IgM antibodies to human cytomegalovirus in the patient specimen. PA1 (a) providing a solid phase containing the fusion protein H10/A1C2/F3 of pp150 of human cytomegalovirus; PA1 (b) introducing a patient specimen to the solid phase of step (a); PA1 (c) introducing peptides A1C2 and F3 of pp150 of human cytomegalovirus to the solid phase of step (a); PA1 (d) allowing IgM antibodies to human cytomegalovirus to specifically bind to the fusion protein H10/A1C2/F3 and the peptides A1C2 and F3; and PA1 (e) determining the level of IgM antibodies to human cytomegalovirus in the patient sample. PA1 (a) microparticles coated with at least a portion of human cytomegalovirus, preferably pp150, more preferably purified recombinant HCMV antigen, suspended in a diluent containing at least one peptide capable of specifically binding IgM antibodies to human cytomegalovirus, preferably the peptides A1C2 and F3; PA1 (b) IgM conjugate in a diluent; and PA1 (c) buffered diluent containing a blocking agent. PA1 (a) a strip of material, preferably a strip made of nitrocellulose, containing HCMV antigens, preferably purified viral and recombinant HCMV antigens; PA1 (b) sample dilution buffer containing at least one peptide capable of specifically binding IgM antibodies to human cytomegalovirus, preferably the peptides A1C2 and F3; PA1 (c) IgM conjugate in a diluent, preferably goat anti-human IgM alkaline phosphatase conjugate in a buffered calf serum diluent; PA1 (d) buffered wash solution; and PA1 (e) color development reagent.
In particular, the aforementioned regions include the amino acid sequences of pp52, pp150, pp65, and pp38 in full, fused together with protein CKS or at least a part thereof.
Finally, U.S. Ser. No. 08/765,856 relates to the use of a mixture of mono- and poly- epitope fusion proteins to detect Cytomegalovirus-specific IgM in human sera by enzyme immunoassay, the mixture being characterized in that it includes, in combination:
U.S. Ser. No. 08/765,856 further relates to the use of the above mixture of fusion proteins to produce a diagnostic kit to detect Cytomegalovirus. specific IgM in human sera by enzyme immunoassay.
The fusion protein, H10/A1C2/F3, was found to have a much higher IgG and IgM reactivity than that obtainable by properly combining in one single test kit the proteins containing the three distinct epitopes. A synergic effect, therefore, takes place, probably owing to the fact that, arranging such epitopes, as are normally far off, close to one another on the same viral protein (A1C2 and F3 of ppUL32, for instance) or even on different proteins (such as H10 of ppUL44), causes conformational epitopes, likely to be present in the original complete viral proteins and/or on the virions, to be "mimicked".
The new fusion protein, comprising the aforementioned sequences, has indeed proven to be as active as the purified virions in binding with IgG and IgM of infected subjects, thus finally allowing production of a standardized antigen, both as to quality and as to quantity, capable of actually replacing purified virions and/or infected cells in the preparation of diagnostic kits for HCMV infection serodiagnosis. Clearly, the previously described epitopes may be included into the new fusion protein as according to the invention in U.S. Ser. No. 08/765,856 in the order of sequence SEQ ID NO: 2, or else in any other sequence, also allowing for bridge sequences to be inserted between them, like that corresponding to nucleotides 757 to 771 of SEQ ID NO: 1.
According to a further aspect of the invention of U.S. Ser. No. 08/765,856, and starting from the results described above, the poly-epitope protein according to the first aspect of the invention has been further studied arranging the said regions in a different way and combining the recombinant material, expressed fused together with protein CKS, with immunogenic regions of other HCMV proteins, so obtaining a mixture of fusion proteins highly effective against IgM.
The fusion proteins according to the invention of U.S. Ser. No. 08/765,856 can be used alone or mixed together, for direct preparation of diagnostic reagents for serological detection of HCMV-specific antibodies, in that their ability of efficiently binding anti-HCMV IgM, in particular those produced during a primary phase of the infection, is substantially 100%. Such reagents may comprise the mixture of fusion proteins according to the invention of U.S. Ser. No. 08/765,856, eventually in combination with other HCMV antigens, or else more complex fusion proteins, including the immunogenic sequences repeated many times and anyway in combination with one another.
The proteic materials represented by the fusion proteins according to the invention of U.S. Ser. No. 08/765,856 can be further used for the preparation, by means of known techniques, of diagnostic reagents for serological detection of human Cytomegalovirus and/or antigens for the detection thereof represented by specific polygonal sera, or by monoclonal antibodies (MaB) directed against the proteins of the invention of U.S. Ser. No. 08/765,856 and obtained via a number of different methods.
These diagnostic reagents, containing the proteins according to the invention of U.S. Ser. No. 08/765,856, can be used, in tum, for the production of diagnostic kits for the demonstration, by serological methods, of the appearance of antibodies to human Cytomegalovirus (HCMV), wherein the reagent is adsorbed on nitrocellulose paper (blotting). Finally, the proteins according to the invention of U.S. Ser. No. 08/765,856 can be used, after being purified, in ELISA assays, latex agglutination tests, RIA, as well as in all known serological screening methods for the presence of HCMV-specific IgG and IgM, both manual and partially or completely automated.
A problem with the assay utilizing the fusion protein H10/A1C2/F3 is that it is so sensitive that low levels of HCMV-specific IgM antibodies that are present in individuals of low risk are frequently detected. In other words, the assay indicates too many false positive results. The diagnosis of recent HCMV infection, e.g., 0 to 8 months, is questionable because the low level of IgM antibody present in individuals experiencing an asymptomatic reactivation from a previous HCMV infection may be detected. If the level of fusion protein H10/A1C2/F3 in the reagent is reduced to improve the specificity of the assay, the sensitivity of the assay is also reduced. In other words, it is undesirable to obtain a positive result in asymptomatic patients who have not had an infection within the previous eight months. Accordingly, it would be desirable to provide an assay for HCMV that not only detects IgM antibody indicative of recent infection, but that also fails to detect IgM antibody that is not indicative of recent infection.